Method of use for murine leukaemia inhibitory factor-binding protein (mLBP)

ABSTRACT

The present invention relates to a isolated leukaemia inhibitory factor (LIF)-binding protein (LBP) in soluble form and obtainable from a first mammalian species, said LBP capable of inhibiting the ability of LIF from a second mammalian species to induce differentiation of M1 myeloid leukaemic cells in vitro to a greater extent when compared to its ability to inhibit LIF from said first mammalian species.

The present invention relates to a Leukaemia Inhibitory Factor-BindingProtein (LBP) and more particularly to a soluble LBP, uses thereof andcompositions containing same.

Leukaemia Inhibitory Factor (LIF) is a polyfunctional glycoprotein withactions on a broad range of tissue and cell types, including inductionof differentiation in a number of myeloid leukaemic cell lines,suppression of differentiation in normal embryonic stem cells,stimulation of proliferation of osteoblasts and DA-1 haemopoietic cellsand potentiation of the proliferative action of interleukin-3 (IL-3) onmegakaryocyte precursors. Functionally, it is able to switch autonomicnerve signalling from adrenergic to cholinergic mode, stimulate calciumrelease from bones, stimulate the production of acute phase proteins byhepatocytes and induce loss of fat deposits by inhibiting lipoproteinlipase-mediated lipid transport into adipocytes¹.

This array of actions is puzzling since it is difficult to conceive ofany situation that would require a coordinated response in all the knowntarget tissues of LIF. Actions of LIF, therefore, are probably designedto be restricted by co-localisation of LIF-producing cells andLIF-responsive cells, with tight regulation of LIF production. However,such an arrangement is likely to result in some release of LIF into thecirculatory system including blood and other bodily fluids.

In work leading up to the present invention, the inventors discovered aLIF-binding protein in serum which is capable of inhibiting thebiological activity of LIF. The identification of this LIF antagonistwill now permit greater control in LIF therapy and to prevent anysystemic effects of locally administered LIF which are nottherapeutically desirable. It also provides a new agent useful in thetreatment of LIF associated diseases or conditions. In a particularembodiment, the inventors have discovered that the inhibitory effect ofthe LIF-binding protein may be more pronounced in heterologous systems,i.e. where the LIF-binding protein from one mammal is used to inhibitLIF in another mammal.

Accordingly, one aspect of the present invention provides a LIF-bindingprotein (LBP) in soluble form and isolatable from a mammal.

More particularly, the present invention is directed to an isolated LBPin soluble form and obtainable from a first mammalian species, said LBPcapable of inhibiting the ability of LIF from a second mammalian speciesto induce differentiation of M1 myeloid leukaemic cells in vitro to agreater extent when compared to its ability to inhibit LIF from saidfirst mammalian species.

The isolated LBP is preferably biologically pure meaning that itrepresents at least 20%, preferably at least 50%, even more preferablyat least 70% and still more preferably at least 85% of the molecule in asolution or composition as determined by weight, biological activity orother convenient means of measurement. Notwithstanding that the LBP isisolated, it may also be in the form of a composition. According to thisaspect of the present invention there is contemplated a compositioncomprising an LBP in soluble form and obtainable from a first mammalianspecies, said LBP capable of inhibiting the ability of LIF from a secondmammalian species to induce differentiation of M1 myeloid leukaemiccells in vitro to a greater extent when compared to its ability toinhibit LIF from said first mammalian species, said compositionsubstantially free of protein molecules not having LBP properties.

The isolated LBP in soluble form and obtainable from the first mammalianspecies is further characterised in that the LBP has at least a 100 foldhigher binding affinity for a LIF from the second mammalian speciescompared to the binding affinity for a LIF from said first mammalianspecies.

In accordance with the present invention, the first mammal is preferablya human, mouse or rat or other rodent, pig, cow, sheep or otherruminant, goat, horse or primate. The second mammal may also be a human,mouse or rat or other rodent, pig, cow, sheep or other ruminant, goat,horse or primate. Preferably, the first mammal is a non-human mammal andthe second mammal is a human. Most preferably, the first mammal is amouse and the second mammal is a human.

Accordingly, in a preferred embodiment, the present invention isdirected to a LBP in soluble form isolatable from a murine animal. Moreparticularly, the present invention provides a LBP in soluble formisolatable from a murine animal, said LBP capable of greater inhibitionof human LIF compared to murine LIF.

The isolated LBP may be the naturally occurring molecule, a naturallyoccurring derivative, part or fragment thereof or may be a recombinantor synthetic form of the molecule including any recombinant or syntheticderivatives, parts or fragments thereof. The LBP may be naturallyglycosylated, partially glycosylated or unglycosylated or may have analtered glycosylation pattern from the naturally occurring molecule. Themolecule may, for example, undergo treatment with N-glycanase resultingin a deglycosylated or substantially deglycosylated molecule. A“derivative” of LBP is considered herein to generally comprise a singleor multiple amino acid insertion, deletion and/or substitution of aminoacid residues relative to the naturally occurring sequence or aninsertion, deletion and/or substitution of molecules associated with LBPsuch as carbohydrate moieties. A “derivative” is also considered to be amolecule with at least 45% amino acid sequence similarity to the aminoacid sequence of the LBP.

Amino acid insertional derivatives of LBP include amino and/or carboxylterminal fusions as well as intra-sequence insertions of single ormultiple amino acids. Insertional amino acid sequence variants are thosein which one or more amino acid residues are introduced into apredetermined site in the protein although random insertion is alsopossible with suitable screening of the resulting product. Deletionalvariants are characterised by the removal of one or more amino acidsfrom the sequence. Substitutional amino acid variants are those in whichat least one residue in the sequence has been removed and a differentresidue inserted in its place. Typical subsitutions are those made inaccordance with the following Table 1:

TABLE 1 Suitable residues for amino acid substitutions Original ResidueExemplary Substitutions Ala Ser Arg Lys Asn Gln; His Asp Glu Cys Ser GlnAsn Glu Ala Gly Pro His Asn; Gln Ile Leu; Val Leu Ile; Val Lys Arg; Gln;Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; PheVal Ile; Leu

Where the LBP is derivatised by amino acid substitution, the amino acidsare generally replaced by other amino acids having like properties, suchas hydrophobicity, hydrophilicity, electronegativity, bulky side chainsand the like. Amino acid substitutions are typically of single residues.Amino acid insertions will usually be in the order of about 1-10 aminoacid residues and deletions will range from about 1-20 residues.Preferably, deletions or insertions are made in adjacent pairs, i.e. adeletion of two residues or insertion of two residues.

The amino acid variants referred to above may readily be made usingpeptide synthetic techniques well known in the art, such as solid phasepeptide synthesis⁽¹⁴⁾ and the like, or by recombinant DNA manipulations.Techniques for making substitution mutations at predetermined sites inDNA having known or partially known sequence are well known and include,for example, M13 mutagenesis. The manipulation of DNA sequences toproduce variant proteins which manifest as substitutional, insertionalor deletional variants are conveniently described, for example, inManiatis et al⁽¹⁵⁾.

Other examples of recombinant or synthetic mutants and derivatives ofthe LBP of the present invention include single or multiplesubstitutions, deletions and/or additions of any molecule associatedwith the LBP such as carbohydrates, lipids and/or proteins orpolypeptides.

In one embodiment, the LBP is truncated at its carboxy terminal endportion to render said LBP soluble.

In another embodiment, the LBP is a fusion molecule between LBPs fromsaid first and second mammalian species.

According to this embodiment, there is provided a fusion polypeptidedefining an LBP, said fusion polypeptide comprising first and secondamino acid sequences wherein said first amino acid sequence is derivablefrom an LBP from a first mammalian species and said second amino acidsequence is derivable from an LBP from a second mammalian specieswherein the LBP from said first mammalian species is capable ofinhibiting the ability of LIF from said second mammalian species toinduce differentiation of M1 myeloid leukaemic cells in vitro to agreater extent when compared to its ability to inhibit LIF from saidfirst mammalian species such that said fusion polypeptide retains theability to inhibit LIF from said second mammalian species to a greaterextent than LIF from said first mammalian species.

In a preferred embodiment, the first mammalian species is a mouse, rator other rodent, pig, cow, sheep or other ruminant, goat, horse orprimate and said second mammalian species is a human. Most preferably,the first mammalian species is a mouse and the fusion polypeptide isreferred to as a “humanised” form of the mouse LBP. Such molecules areparticularly advantageous in avoiding or reducing possible induction ofan antigenic immune response by administering to said first mammal, anLBP from said second mammal.

According to this most preferred aspect of the present invention, thereis provided a fusion polypeptide defining an LBP, said fusionpolypeptide comprising first and second amino acid sequences whereinsaid first amino acid sequence is derivable from an LBP from a mouse andsaid second amino acid sequence is derivable from an LBP from a humanwherein said fusion polypeptide is capable of inhibiting the ability ofLIF from a human to induce differentiation of M1 myeloid leukaemic cellsin vitro to a greater extent when compared to its ability to inhibitmouse LIF and wherein administration of said fusion polypeptides into ahuman results in a substantially reduced immune response against saidfusion polypeptide compared to the administration to said human ofnative or recombinant mouse LBP. Conveniently, an “immune response” ismeasured by titre of antibodies specific to a molecule and/or involveextent of a cellular immune response.

The fusion polypeptides can be prepared by a range of suitable methodsbut conveniently is by a method similar to the method employed by theinventors to map the site on the hLIF molecule that confers both bindingto the hLIF receptor α-chain and the unusual high affinity binding tothe mouse LIF receptor α-chain (mLBP) similar to that described in theexperiment summarised in FIG. 9. In particular, a hLBP molecular framework is used to construct a series of mouse-human mLBP chimaericmolecules in order to determine the minimum number of hLIF amino acidresidues that is necessary to substitute into the hLBP sequence in orderto create a molecule that has the properties that are peculiar to hLBP.

Reference in the specification and claims herein to “LBP1” includesreference to a fusion polypeptide defining an LBP as defined above.

The terms “analogues” and “derivatives” also extend to any functionalchemical equivalent of the LBP characterised by its increased stabilityand/or efficacy in vivo or vitro. The terms “analogues” and“derivatives” also extend to any amino acid derivative of the LBP asdescribed above.

Analogues of LBP contemplated herein include, but are not limited to,modifications to side chains, incorporation of unnatural amino acidsand/or derivatising the molecule and the use of crosslinkers and othermethods which impose conformational constraints on the LBP molecule orits analogues. Examples of side chain modifications contemplated by thepresent invention include modifications of amino groups such as byreductive alkylation by reaction with an aldehyde followed by reductionwith NaBH₄; amidiation with methylacetimidate; acylation with aceticanhydride; carbamoylation of amino groups with cyanate;trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzenesulphonic acid (TNBS); acylation of amino groups with succinic anhydrideand tetrahydrophthalic anhydride; and pyridoxylation of lysine withpyridoxal-5′-phosphate followed by reduction with NaBH₄.

The guanidino group of arginine residues may be modified by theformation of heterocyclic condensation products with reagents such as2,3- butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation viaO-acylisourea formation followed by subsequent derivitisation, forexample, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylationwith iodoacetic acid or iodoacetamide; performic acid lidation tocysteic acid; formation of a mixed disulphides with other thiolcompounds; reaction with maleimide, maleic anhydride or othersubstituted maleimide; formation of mercurial derivatives using4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid,phenylmercury chloride, 2-chloromercuri-4-nitrophenol and othermercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation withN-bromosuccinimide or alkylation of the indole ring with2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residueson the other hand, may be altered by nitration with tetranitromethane toform a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may beaccomplished by alkylation with iodoacetic acid derivatives orN-carbethoxylation with diethylpyrocarbonate. Other types ofmodifications include iodination of tyrosine and biotinylation oflysine.

Examples of incorporating unnatural amino acids and derivatives duringprotein synthesis include, but are not limited to, use of norleucine,4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid,6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine,ornithine, sarcosine, 4-amino-3-hydroxy6-methylheptanoic acid, 2-thienylalanine and/or D-isomers of amino acids.

Crosslinkers can be used, for example, to stabilise 3D conformations,using homo bifunctional crosslinkers such as the bifunctional imidoesters having (CH₂)_(n) spacer groups with n=1 to n=6, glutaraldehyde,N-hydroxysuccinimide esters and hetero-bifunctional reagents whichusually contain an amino-reactive moiety such as N-hydroxysuccinimideand another group-specific reactive moiety such as maleimido or dithiomoiety (SH) or carbodiimide (COOH). In addition, peptides could beconformationally constrained by, for example, incorporation of C_(α) andN₆₀-methylamino acids, introduction of double bonds between C_(α) andC_(β) atoms of amino acids and the formation of cyclic peptides oranalogues by introducing covalent bonds such as forming an amide bondbetween the N and C termini, between two side chains or between a sidechain and the N or C terminus.

The LBP molecule of the present invention may also be pegolated usingpolyethylene glycol (PEG) or other equivalent or similar fatty acid toaid in increasing stability and/or the half life of the protein. In thisregard, reference can conveniently be made, for example, to U.S. Pat.No. 5,089,261 which describes the use of PEG to increase the in vivohalf life of interleukin 1.

Conveniently, the LBP may be in a purified or semi-purified form fromblood, serum or other biological fluid sample. More conveniently, theLBP can be purified or semi-purified by sequential fractionation usingaffinity chromatography on an immobilised LIF column, anion exchangechromatography, size exclusion chromatography and preparative nativepolyacrylamide gel electrophoresis. One or more of the foregoing stepsmay be altered or a similar or equivalent step substituted thereforwithout departing from the scope of the present invention provided theLBP is enriched from a particular biological sample.

The LIF used in the LIF affinity column is generally of first mammalianorigin, i.e. same mammalian or gin for both LBP and LIF although any LIFcapable of binding the LBP to be purified can be used in the affinitycolumn.

Conveniently, the purification of the LBP is monitored by binding tolabelled LIF and preferably radioactively labelled LIF, such as usingknown ¹²⁵I-LIF binding assays. Other means of monitoring LBP activitycan also be used, such as specific antibody binding or inhibition of LIFactivity through competitive assays.

The murine LBP (mLBP) in accordance with the preferred aspects of thepresent invention when purified as generally described above has anapparant molecular weight as determined on SDS-PAGE of approximately90,000±10,000 daltons in glycosylated form and 65,000±10,000 daltons inanother glycosylated form and specifically binds ¹²⁵I-murine LIF (mLIF)with an equilibrium dissociation constant of about 0.5-2 nM.Furthermore, mLIF is approximately 1,000-10,000-fold less effective thanhuman LIF (hLIF) in competing with ¹²⁵I-hLIF for binding to mLBP. Moresignificantly, however, as shown by the in vitro effects hereindescribed, mLBP is at least 100-fold and is generally about 1,000-foldmore active as an inhibitor of hLIF than of mLIF. In addition, thedirect binding affinity of mLBP for hLIF is approximately 100 timeshigher than that for mLIF.

In the most preferred embodiment of the present invention, the mLBP hasan amino acid sequence in the N-terminal region comprisingGly-Val-Gln-Asp-Leu-Lys-Cys-Thr-Thr-Asn-Asn-Met-Arg-Val-Trp-Asp-Cys-Thr-Trp-Pro-Ala-Pro-Leu (SEQ ID No. 1), is soluble, particularly in aqueous bufferedsolutions and has an apparant molecular weight in the glycosylated formof 90,000±10,000 daltons, and preferably 90,000±5,000 daltons asdetermined by SDS-PAGE. On the basis of treatment with N-glycanase, onedeglycosylated form has a molecular weight of approximately65,000±15,000 daltons and preferably 65,000±10,000 daltons and anotherdeglycosylated form has a molecular weight of approximately50,000±10,000 daltons as determined by SDS-PAGE. The present inventionextends to LBP molecules having an N-terminal amino acid sequence withat least 45%, preferably at least 55%, more preferably at least 65% andstill more preferably at least 75-85% and even more preferably greaterthan 90% similarity to the amino acid sequence:Gly-Val-Gln-Asp-Leu-Lys-Cys-Thr-Thr-Asn-Asn-Met-Arg-Val-Trp-Asp-Cys-Thr-Trp-Pro-Ala-Pro-Leu(SEQ ID No. 1).

The present invention extends to nucleic acid molecules and preferablyisolated nucleic acid molecules comprising a sequence of nucleotidesencoding or complementary to a sequence encoding an LBP as hereinbeforedescribed including a fusion polypeptide defining an LBP. The nucleotidesequence may correspond to the naturally occurring amino acid sequenceof the LBP or may contain single or multiple nucleotide substitutions,deletions and/or additions thereto. Preferably, the nucleic acid encodesan LBP with an N-terminal amino acid sequence comprisingGly-Val-Gln-Asp-Leu-LysCys-Thr-Thr-Asn-Asn-Met-Arg-Val-Trp-Asp-Cys-Thr-Trp-Pro-Ala-Pro-Leu (SEQID No. 1) or contains a nucleotide sequence capable of hybridising underlow, preferably under medium and more preferably under high stringencyconditions to a nucleotide sequence encoding the above stated amino acidsequence. Put in alternative terms the hybridising nucleotide sequenceis at least 45%, preferably at least 55%, more preferably at least65-75% and even more preferably greater than 85% similar to thenucleotide sequence encoding the above-stated amino acid sequence.

For the purposes of defining the levels of stringency, reference canconveniently be made to Sambrook et al⁽¹⁵⁾ at pages 387-389 where thewashing step at paragraph 11 is considered herein to be high stringency.A low stringency wash is defined herein to be 0.1%-0.5% w/v SDS at37-45° C. for 2-3 hours and a medium level of stringency is consideredherein to be 0.25%-5% w/v SDS at ≧45° C. for 2-3 hours. The alternativeconditions are applicable depending on concentration, purity and sourceof nucleic acid molecules.

In a particularly preferred embodiment, the nucleic acid molecule of thepresent invention is in an expression vector capable of replication andexpression in eukaryotic organisms (e.g. CHO cells or other mammaliancells, yeast cells, insects cells) and/or in prokaryotic organisms (e.g.E. coli). Such expression vectors and cells transformed with same areconvenient sources of the recombinant LBP molecules of the presentinvention.

The LBP of the present invention will be particularly useful as aninhibitor of the systemic effects of locally produced or administeredLIF. Where the use of a heterologous LBP relative to the mammal to betreated is significantly more active than homologous LBP (i.e. LBP fromthe same species of mammal), then this high activity is particularlyadvantageous in reducing, for example, the immunological consequences ofintroducing the heterologous protein into a mammal. The high activitywill also enable the administration of as little LBP as possible toensure that the LBP can be localised to a particular site and cannotdisseminate to other areas of the mammal. It may also be important inconjunction with LIF therapy to maintain effective levels of LBP in thecirculatory fluids including serum so as to prevent dissemination of LIFadministered in the course of the therapy, such as where LIF is locallyadministered.

Accordingly, another aspect of the present invention contemplates amethod of inhibiting the activity of LIF in a mammal comprisingadministering to said mammal, an effective amount of a soluble LBP.

More particularly, the present invention contemplates a method ofinhibiting the activity of LIF in a mammal comprising administering tosaid mammal, an effective amount of a soluble heterologous LBP whereinsaid heterologous LBP is capable of greater inhibition of the LIF in themammal to be treated when compared to a LIF of same mammalian origin tothe LBP.

Preferably, the mammal to be treated is a human and the LBP is mLBP.

Preferably, this method is used in the inhibition of the systemiceffects of locally produced LIF which are therapeutically undesirable,unintended or unwanted.

Administration may be by any convenient means applicable to thecondition being treated but is particularly conveniently administeredlocally to the site where LIF is to be inhibited. Alternatively, the LBPmay be administered to elevate serum levels while LIF is administeredlocally.

The effective amount of LBP will vary depending on the mammal andcondition to be treated but car, range from serum levels of 0.001 μg/mlto 100 μg/ml, preferably 0.01 μg/ml to 50 μg/ml, more preferably 0.1μg/ml to 20 μg/ml and most preferably 0.5 μg/ml to 10 μg/ml. The amountrequired will be that amount required to completely or partially inhibitLIF activity or at least reduce it to a clinically acceptable level.Furthermore, the “LIF activity” may be all activities associated withLIF or only some of these activities and may be measured in any numberof convenient ways such as in a bioassay⁽⁹⁾ or a receptor-binding assay.

The LBP may be administered alone or in combination with other activecompounds such as, but not limited to, cytokines, antibiotics,anti-cancer agents or immuno-stimulatory or reducing compounds.Administration of the LBP and other active compounds may be bysimultaneous or sequential administration. Furthermore, whether the LBPis administered alone or in combination with other compounds, a singledose of LBP may be sufficient or multiple doses or continuous infusionmay be required depending on the condition and mammal to be treated andwhether any adverse clinical reactions appear.

Yet a further aspect of the present invention provides a pharmaceuticalcomposition comprising LBP as hereinbefore defined and one or morepharmaceutically acceptable carriers and/or diluents. The preparation ofpharmaceutical composition is discussed generally in Remington'sPharmaceutical Sciences, 17th ed. Mach Publishing Co., Easton, Pa., USA.Alternatively, the LBP may be administered genetically using transgenicanimal cells or microbial cells.

The active ingredients of the pharmaceutical composition comprising anLBP as herein described are contemplated to exhibit excellent activitywhen administered in a dosage regimen adjusted to provide the optimumtherapeutic response. For example, several divided doses may beadministered daily, weekly, monthly or in other suitable time intervalsor the dose may be proportionally reduced as indicated by the exigenciesof the situation. The active compound may be administered in aconvenient manner such as by the oral, topical, intravenous,intramuscular, subcutaneous, intranasal, intradermal or suppositoryroutes or implanting (eg using slow release molecules). Depending on theroute of administration, the active ingredient which comprises an LBPmay be required to be coated in a material to protect said ingredientfrom the action of enzymes, acids and other natural conditions which mayinactivate said ingredients. In order to administer the vaccine by otherthan parenteral administration, the LBP may be coated by, oradministered with, a material to prevent its inactivation. For example,the LBP may be administered in an adjuvant, co-administered with enzymeinhibitors or in liposomes.

The active compound may also be administered in dispersions prepared inglycerol, liquid polyethylene glycols, and/or mixtures thereof and inoils. Under ordinary conditions of storage and use, these preparationscontain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases the form must be sterile and mustbe fluid to the extent that easy syringability exists. It must be stableunder the conditions of manufacture and storage and must be preservedagainst the contaminating action of microorganisms such as bacteria andfungi. The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thormerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by, for example, theuse in the compositions of agents delaying absorption.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredient(s) into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and the freeze-dryingtechnique which yield a powder of the active ingredient plus anyadditional desired ingredient from previously sterile-filtered solutionthereof.

When the LBP is suitably protected as described above, the molecule maybe orally administered, for example, with an inert diluent or with anassimilable edible carrier, or it may be enclosed in hard or soft shellgelatin capsule, or it may be compressed into tablets, or it may beincorporated directly with the food of the diet. For oral therapeuticadministration, the active compound may be incorporated with excipientsand used in the form of ingestible tablets, buccal tablets, troches,capsules, elixirs, suspensions, syrups, wafers, and the like. Suchcompositions and preparations should contain at least 1% by weight ofactive compound. The percentage of the compositions and preparationsmay, of course, be varied and may conveniently be between about 5 toabout 80% of the weight of the unit. The amount of active compound inthe vaccine compositions is such that a suitable dosage will beobtained. Preferred compositions or preparations according to thepresent invention are prepared so that an oral dosage unit form containsbetween about 0.5 ug and 20 mg of active compound.

The tablets, troches, pills, capsules and the like may also contain thefollowing: a binder such as gum gragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such asucrose, lactose or saccharin may be added or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavouring. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier. Various other materials may be present ascoatings or to otherwise modify the physical form of the dosage unit.For instance, tablets, pills, or capsules may be coated with shellac,sugar or both. A syrup or elixir may contain the active compound,sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavoring such as cherry or orange flavor. Ofcourse, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compound may be incorporated intosustained-release preparations and formulations.

As used herein, pharmaceutically acceptable carriers and/or diluentsinclude any and all solvents, dispersion media, aqueous solutions,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like. The use of such media and agents forpharmaceutical active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, use thereof in the composition is contemplated.Supplementary active ingredients can also be incorporated into thecompositions.

To reduce the potentially disadvantageous effects of administering aheterologous LBP to a mammal, it may be possible to derivatise orotherwise alter the heterologous LBP to reduce its antigenicity in themammal to be treated. This can be accomplished by rendering the LBP morelike a protein from the mammal to be treated. For example, the LBP canbe coupled or masked with a protein or polypeptide or other suitablemolecule from the species of mammal to be treated or coupling or fusingthe heterologous LBP or parts thereof to the LBP from the targetspecies. In a particularly preferred embodiment, the murine LBP isrendered non-immunogenic in a human and has been derivativatised morelike a human molecule.

In yet another aspect of the present invention, the LBP of firstmammalian origin is used to detect LIF of second mammalian origin.

In one embodiment, the method contemplated for detecting LIF in abiological sample said method comprising contacting said biologicalsample with an immobilised LBP from a mammal wherein the LBP is capableof inhibiting the ability of the first mentioned LIF to inducedifferentiation of M1 myeloid leukaemic cells in vitro to a greaterextent when compared to its ability to inhibit LIF of same mammalianorigin as LBP and/or wherein said LBP has at least a 100-fold higherbinding affinity for said first mentioned LIF compared to the bindingaffinity of LIF of same mammalian origin as said LBP wherein saidcontact is for a time and under conditions sufficient for a complex toform between the immobilised LBP and the LIF in the sample; contactingthe LBP-LIF complex with an antibody specific for said LIF and labelledwith a reporter molecule capable of providing a signal; determining thepresence of bound LIF on the basis of the signal produced by saidreporter molecule.

In an alternative embodiment, the LBP-LIF complex is contacted with anunlabelled antibody specific to said LIF and then LIF is detected by asecond antibody labelled with a reporter molecule and specific to saidfirst antibody.

In yet another alternative embodiment, the LIF in the sample isimmobilised (e.g. by an immobilised antibiody) and then bound LIFdetected by labelled LBP or first by unlabelled LBP followed by alabelled antibody specific to said LBP.

An embodiment of this aspect of the present invention is describedhereinafter with reference to the preferred embodiment of using mLBP todetect hLIF. The present invention, however, is not so limited andextends to the use of LBP and LIF from other mammals.

According to this embodiment, there is contemplated a method ofdetecting hLIF in a biological sample, said method comprising contactingsaid sample to mLBP as hereinbefore defined, immobilised to a solidsupport for a time and under conditions sufficient for a mLBP-hLIFcomplex to form and then detecting for the presence of said mLBP-hLIFcomplex.

In a particularly preferred method, the mLBP-hLIF complex is detected bycontacting the complex with an antibody for hLIF with the antibodyitself being labelled with a reporter molecule or with an additionalstep of contacting the mLBP-hLIF-antibody complex with a labelled secondantibody capable of binding to the first antibody.

The antibodies may be polyclonal or monoclonal and both are obtainableby immunization of a suitable animal with hLIF and either type isutilizable in the LIF assay. The methods of obtaining both types of seraare well known in the art. Polyclonal sera are less preferred but arerelatively easily prepared by injection of a suitable laboratory animalwith an effective amount of hLIF, or antigenic parts thereof, collectingserum from the animal, and isolating specific sera by any of the knownimmunoadsorbent techniques. Although antibodies produced by this methodare utilizable in virtually any type of LIF assay, they are generallyless favoured because of the potential heterogeneity of the product.

The use of monoclonal antibodies in an immunoassay is particularlypreferred because of the ability to produce them in large quantities andthe homogeneity of the product. The preparation of hybridoma cell linesfor monoclonal antibody production derived by fusing an immortal cellline and lymphocytes sensitized against the immunogenic preparation canbe done by techniques which are well known to those who are skilled inthe art. (See, for example Douillard and Hoffman, Basic Facts aboutHybridomas, in Compendium of Immunology Vol II, ed. by Schwartz, 1981;Kohler and Milstein, Nature 256: 495-499, 1975; European Journal ofImmunology 6: 511-519, 1976).

The presence of a hLIF may be accomplished in a number of ways such asby Western blotting and ELISA procedures. A wide range of immunoassaytechniques are available as can be seen by reference to U.S. Pat. Nos.4,016,043, 4,424,279 and 4,018,653. These, of course, include bothsingle-site and two-site or “sandwich” assays of the non-competitivetypes, as well as in the traditional competitive binding assays. Theseassays also include direct binding of a labelled antibody to a target.

Sandwich assays are among the most useful and commonly used assays andare favoured for use in the present invention. A number of variations ofthe sandwich assay technique exist, and all are intended to beencompassed by the present invention. Briefly, in a typical forwardassay, mLBP is immobilized on a solid substrate and the sample to betested brought into contact with the bound molecule. After a suitableperiod of incubation, for a period of time sufficient to allow formationof an mLBP-hLIF complex, an antibody specific to the hLIF, labelled witha reporter molecule capable of producing a detectable signal, is thenadded and incubated allowing sufficient time for the formation of acomplex of mLBP-hLIF-labelled antibody. Any unreacted material is washedaway, and the presence of the hLIF is determined by observation of asignal produced by the reporter molecule. The results may either bequalitative, by simple observation of the visible signal, or may bequantitated by comparing with a control sample containing known amountsof hapten. Variations on the forward assay include a simultaneous assay,in which both sample and labelled antibody are added simultaneously tothe bound mLBP. In accordance with the present invention the sample isone which might contain hLIF and includes biological fluid (e.g. blood,serum, tissue extract) fermentation fluid and supernatant fluid such asfrom a cell culture.

In the typical forward sandwich assay, mLBP or a hLIF-binding partthereof is either covalently or passively bound to a solid surface. Thesolid surface is typically glass or a polymer, the most commonly usedpolymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinylchloride or polypropylene. The solid supports may be in the form oftubes, beads, discs of microplates, or any other surface suitable forconducting an immunoassay. The binding processes are well-known in theart and generally consist of cross-linking covalently binding orphysically adsorbing mLBP to the polymer. After immobilising the mLBP,the polymer-mLBP complex is washed in preparation for the test sample.An aliquot of the sample to be tested is then added to the solid phasecomplex and incubated for a period of time sufficient (e.g. 2-40minutes) and under suitable conditions (e.g. 25° C.) to allow binding ofhLIF in the sample to the immobilised mLBP. Following the incubationperiod, the complex is washed and optionally dried and then incubatedwith an antibody specific for hLIF. The antibody is linked to a reportermolecule which is used to indicate the binding of hLIF to mLBP.Alternatively, a second antibody conjugated to a reporter molecule andcapable of binding to the first antibody may be used.

An alternative method involves immobilizing the target molecules (i.e.hLIF) in the biological sample and then exposing the immobilized targetto mLBP which may or may not be labelled with a reporter molecule.Depending on the amount of target and the strength of the reportermolecule signal, a bound target may be detectable by direct labelling ofmLBP. Alternatively, a labelled antibody, specific to mLBP is exposed tothe complex to form a tertiary complex. The complex is detected by thesignal emitted by the reporter molecule.

By “reporter molecule” as used in the present specification, is meant amolecule which, by its chemical nature, provides an analyticallyidentifiable signal which allows the detection of antigen-boundantibody. Detection may be either qualitative or quantitative. The mostcommonly used reporter molecules in this type of assay are eitherenzymes, fluorophores or radionuclide containing molecules (i.e.radioisotopes) and chemiluminescent molecules.

In the case of an enzyme immunoassay, an enzyme is conjugated to thehLIF-specific antibody, generally by means of glutaraldehyde orperiodate. As will be readily recognized, however, a wide variety ofdifferent conjugation techniques exist, which are readily available tothe skilled artisan. Commonly used enzymes include horseradishperoxidase, glucose oxidase, beta-galactosidase and alkalinephosphatase, amongst others. The substrates to be used with the specificenzymes are generally chosen for the production, upon hydrolysis by thecorresponding enzyme, of a detectable color change. Examples of suitableenzymes include alkaline phosphatase and peroxidase. It is also possibleto employ fluorogenic substrates, which yield a fluorescent productrather than the chromogenic substrates noted above. Generally, theenzyme-labelled antibody is added to the mLBP-hLIF complex, allowed tobind, and then the excess reagent is washed away. A solution containingthe appropriate substrate is then added to the tertiary complex. Thesubstrate will react with the enzyme linked to the antibody, giving aqualitative visual signal, which may be further quantitated, usuallyspectrophotometrically, to give an indication of the amount of haptenwhich was present in the sample. “Reporter molecule” also extends to useof cell agglutination or inhibition of agglutination such as red bloodcells on latex beads, and the like.

Alternately, fluorescent compounds, such as fluorecein and rhodamine,may be chemically coupled to antibodies without altering their bindingcapacity. When activated by illumination with light of a particularwavelength, the fluorochrome-labelled antibody adsorbs the light energy,inducing a state to excitability in the molecule, followed by emissionof the light at a characteristic color visually detectable with a lightmicroscope. As in the EIA, the fluorescent labelled antibody is allowedto bind to the mLBP-hLIF. After washing off the unbound reagent, thetertiary complex is then exposed to the light of the appropriatewavelength and the fluorescence observed indicates the presence of thehapten of interest. Immunofluoresence and EIA techniques are both verywell established in the art and are particularly preferred for thepresent method. However, other reporter molecules, such as radioisotope,chemiluminescent or bioluiminescent molecules, may also be employed. Theabove considerations apply where the antibody is an anti-immunoglobulinand is labelled so that a quaternay complex is obtained.

Furthermore, the mLBP of the present invention may be packaged in kitform, for example, to conduct an assay for LIF. The kit is incompartmental form adapted to contain mLBP and may further comprise inthe same or different compartments the reagents for the LIF assay.

The foregoing description is equally applicable to the use of LBP from afirst mammalian species to detect LIF from a second mammalian species ashereinbefore defined. Furthermore, variations such as binding LIF usingimmobilised antibodies and then detecting the bound LIF with LBPconjugated to a reporter molecule or using first LBP then LBP-bindingantibody conjugated to a reporter molecule.

The present invention is further described by reference to the followingnon-limiting Figures and/or Example.

In the Figures:

FIGS. 1A-1B is a graphical representation showing size exclusion profileof ¹²⁵I-mLIF in the presence and absence of normal mouse serum. TheUltrogel AcA44 (20×1 cm) column was equilibrated and run in 20 mM Naphosphate buffer pH 7.4, 150 mM NaCl (PBS) at 0.2 ml/min, collecting 1min fractions that were counted in a gamma counter. (A) 100,000 cpm¹²⁵I-mLIF in 100 μl PBS (B) 100,000 cpm ¹²⁵I-mLIF preincubated with 100μl normal mouse serum for 16 hr at room temperature.

FIGS. 2A-2C are graphical representation showing representativefractionations of normal mouse serum by LIF affinity chromatography,anion exchange chromatography and size exclusion chromatography.Absorbance and specific ¹²⁵I-mLIF binding of fractions in a Con-ASepharose binding assay are shown. (A) 50 ml mouse serum was applied toa column of LIF-pABAE-Sepharose 4B in PBS and eluted with guanidine-HClas described in the Example. (B) Active fractions from affinitychromatography were pooled, applied to a Mono-Q HR 5/5 and eluted with asalt gradient as described in the Example. (C) Active fractions fromanion-exchange chromatography were pooled, concentrated to 100 μl andinjected onto a Superose-12 10/30 column equilibrated in PBS, andelution was carried out isocratically using the same buffer.

FIGS. 3A-3B is a photographic and graphical representation ofpreparative native gel electrophoresis of LBP. Upper panel (A):Fractions from size-exclusion chromatography containing LIF-bindingactivity were pooled and applied to a 20 ml 7.5% w/v polyacrylamidenative gel and electrophoresed in a BioRad Model 491 Prep Cell asdescribed in the Example. Specific ¹²⁵I-mLIF binding of eluted fractionsin a Con-A Sepharose binding assay is shown. Lower panel (B):NaDodSO₄-PAGE of 20 μl aliquots of pooled fractions containing LBPactivity from various stages of purification: s, low molecular weightstandards (Pharmacia) (MW×10⁻³); a, normal mouse serum (1/50); b & c,1^(st) LIF-pABAE Sepharose 4B column; d, 2^(nd) LIF-pABAE Sepharose 4Bcolumn; e, Mono-Q HR 5/5 column; f, Superose-12 10/30 column; g,preparative native gel electrophoresis, arrow shows band of interest.

FIGS. 4A-4C is a graphical representation showing Scatchard analysis of¹²⁵I-mLIF binding to LBP, solubilized murine liver membranes and 3T3-L1cells. (A) Saturation isotherm for ¹²⁵I-mLIF binding to mouse serum (5μl in 210 μl total) showing total cpm bound (▴), non-specific cpm bound() and specific cpm bound (◯). (B) Scatchard transformation of¹²⁵I-mLIF binding to LBP in mouse serum (5 μl in 210 μl total) (◯) andpurified LBP (1 μl in 90 μl total) (). (C) Scatchard transformation of¹²⁵I-mLIF binding to solubilized murine liver membranes (25 μl in 175 μltotal) (◯) and 3T3-L1 cells (0.6×10⁶ in 80 μl total) ().

FIGS. 5A-5B is a graphical representation of displacement curves forunlabelled mLIF (◯) and hLIF () competing with the binding of (A)¹²⁵I-mLIF and (B) ¹²⁵I-hLIF for purified LBP (100 μl total volume).

FIGS. 6A-6B is a graphical representation showing blocking of inductionof differentiation of mLIF stimulated colonies of M1 leukemic cells byaffinity-purified LBP. (A) Titration of LBP in the presence of constantamounts of mLIF as indicated. (B) Titration of mLIF with constant levelsof LBP as indicated.

FIGS. 7A-7B is a graphical representation showing the blocking ofinduction of mLIF and hLIF stimulated colonies of M1 leukaemic cells bynormal mouse serum. mLIF and hLIF have an equal ability to stimulate thedifferentiation of murine M1 colonies. A. Effect on 100 U E. coliderived murine LIF (emLIF) and 800 U E coli derived human LIF (ehLIF).B. Effect on 400 U emLIF and 200 U each of ehLIF, CHO cells derivedhuman LIF (chohLIF) and yeast derived human LIF (YhLIF). As in FIG. 6A,a 1:8 dilution of normal mouse serum (equivalent to 125 ng/ml LBP) wasable to inhibit 50% of activity stimulated by 100 U/ml mLIF. Incontrast, as little as 1:512 dilution of normal mouse serum (equivalentto −1.5 ng/ml LBP) was able to inhibit 50% of the activity stimulated by800 U/ml hLIF.

FIGS. 8A-B is a graphical representation showing specific binding of¹²⁵I-mLIF (A) and ¹²⁵I-hLIF (B) to mLBP immobilized onto 96-well PVCplates. Affinity purified mLBP was diluted in various buffers (PBS, 0.1MNaHCO₃ pH9.5 and 0.1M NaHCO₃ pH9.5 containing 4 μg/ml bovine serumalbumin (BSA)) and 100 μl aliquots incubated in 96-well PVC plates(Costar) for 16 hours at room temperature. The plates were then washedwith PBS containing 0.05% v/v Tween-20 (wash buffer) and blocked withHepes-buffered RPMI medium containing 10% v/v Foetal calf serum (HRF)for 1 hour at room temperature. The plates were again washed with washbuffer then incubated with 50 μl HRF containing ¹²⁵I-mLIF or ¹²⁵I-hLIFfor 16 hours at room temperature. Non-specific binding was determinedfrom incubations containing in addition 20 μl of 50 μg/ml unlabelledmLIF or hLIF. Bound and free labelled ligand were separated by washingthe plates three times in wash buffer. The plates were dried and theassay plates containing the bound labelled ligand were exposed to aphosphorimager screen (Molecular Dynamics) for 24 hours. Results wereanalysed using Imagequant version 3 (Molecular Dynamics) software. Thealphabetically labelled bars are as follows:

a) 5.76 μg/ml mLBP in PBS

b) 2.88 μg/ml mLBP in 0.1M Na bicarbonate pH9.5

c) 1.44 μg/ml mLBP in 0.1M Na bicarbonate pH9.5

d) 0.72 μg/ml mLBP in 0.1M Na bicarbonate pH9.5

e) 0.36 μg/ml mLBP in 0.1M Na bicarbonate pH9.5

f) 0.18 μg/ml mLBP in 0.1M Na bicarbonate pH9.5

g) 5.76 μg/ml mLBP in PBS+4 μg/ml BSA

h) 2.88 μg/ml mLBP in 0.1M Na bicarbonate pH9.5+4 μg/ml BSA

i) 1.44 μg/ml mLBP in 0.1M Na bicarbonate pH9.5+4 μg/ml BSA

j) 0.72 μg/ml mLBP in 0.1M Na bicarbonate pH9.5+4 μg/ml BSA

k) 0.36 μg/ml mLBP in 0.1M Na bicarbonate pH9.5+4 μg/ml BSA

l) 0.18 μg/ml mLBP in 0.1M Na bicarbonate pH9.5+4 μg/ml BSA

m) 5.76 μg/ml mLBP in PBS

n) 2.88 μg/ml mLBP in 0.1M Na bicarbonate pH9.5

o) 1.44 μg/ml mLBP in 0.1M Na bicarbonate pH9.5

p) 0.72 μg/ml mLBP in 0.1M Na bicarbonate pH9.5

q) 0.36 μg/ml mLBP in 0.1M Na bicarbonate pH9.5

r) 0.18 μg/ml mLBP in 0.1M Na bicarbonate pH9.5

s) 5.76 μg/ml mLBP in PBS+4 μg/ml BSA

t) 2.88 μg/ml mLBP in 0.1M Na Bicarbonate pH9.5+4 μg/ml BSA

u) 1.44 μg/ml mLBP in 0.1M Na Bicarbonate pH9.5+4 μg/ml BSA

v) 0.72 μg/ml mLBP in 0.1M Na Bicarbonate pH9.5+4 μg/ml BSA

w) 0.36 μg/ml mLBP in 0.1M Na Bicarbonate pH9.5+4 μg/ml BSA

x) 0.18 μg/ml mLBP in 0.1M Na Bicarbonate pH9.5+4 μg/ml BSA

FIG. 9 is a graphical representation showing competitive binding ofmouse LIF () human LIF (∘), porcine LIF (▴) and a series of mouse-humanLIF chimeric molecules (▪, □, ♦, ⋄) Aliquots of 20 μl of normal mouseserum diluted 1 in 20 in PBS were added to 96-well filtration assayplates containing a 0.65 micron Durapore membrane (Millipore) with 10 μl¹²⁵I-hLIF, 50 μl unlabelled ligand diluted in PBS and 25 μlConcanavalin-A Sepharose 4B (diluted 1 in 4 in 0.1M sodium acetate pH6.0containing 1 mM each MgCl₂, MnCl₂ and CaCl₂) and incubated at roomtemperature, overnight, with agitation. Bound and free radioactivitywere separated by vacuum filtration of the supernatant, and theConcanavalin-A Sepharose pellet was washed once with cold PBS. Assayplates containing the Concanavalin-A Separose pellet were exposed to aphosphorimager screen (Molecular Dynamics) for 24 hours. Results wereanalysed using Imagequant version 3 (Molecular Dynamics) software.

FIG. 10A-C is a graphical representation showing comparative bindingdata for mLIF and hLIF to mLBP and recombinant hLIF receptor α-chain.

(A) Scatchard analysis of ¹²⁵I-mLIF binding to mLBP (normal mouse serumdiluted 1 in 20) (, K_(d)=1-4 nM), ¹²⁵I-hLIF binding to mLBP (normalmouse serum diluted 1 in 1000) (◯, K_(d)=17 pM), ¹²⁵I-hLIF binding toconditioned medium collected 4 days after transfection of COS cells witha plasmid encoding a soluble truncated form of the hLIF receptor α-chain(▴, K_(d)=300-400 pM) and ¹²⁵I-hLIF binding to 44×10⁶ cells/ml Allen1cells, which express a high affinity hLIF receptor (Δ, K_(d)=80 pM).

(B) Competition of unlabelled mouse LIF (, ID₅₀=500 nM) and human LIF(◯, ID₅₀=0.1 nM) with ¹²⁵I-hLIF for binding to mLBP. Experimentalconditions were as described in FIG. 5.

(C) Competition of unlabelled mouse LIF (, ID₅₀>10,000 nM) and humanLIF (◯, ID₅₀=2 nM) with ¹²⁵I-hLIF for binding to conditioned mediumcollected 4 days after transfection of COS cells with a plasmid encodinga soluble truncated form of the hLIF receptor α-chain. Experimentalconditions were as described in FIG. 5.

FIG. 11A is a photographic representation showing Coomassie Blue stainedgel of elution fractions from hLIF-Affigel-10 column. The hLIF-Affigelcolumn was synthesized using 5 ml Affigel-10 (BioRad), 3.5 mg hLIF and 5mg ovalbumin as a filler protein according to manufacturer'sinstructions. An amount of 60 ml of normal C₃H/HeJ mouse serum (as asource of mLBP) was applied to the 5 ml hLIF-Affigel-10 columnequilibrated in PBS. The column was eluted with 85 ml equilibrationbuffer and a 30 ml linear gradient to 6M guanidine-HCl in the samebuffer. Fractions of 2.5 ml were collected throughout at a flow rate of0.5 ml/min, exchanged where necessary into equilibration buffer usingPD10 columns (Pharmacia) and concentrated to 1 ml using Centriconmicroconcentrators (Amicon). SDS-PAGE was performed according to themethod of Laemmli⁽⁴⁾. Aliquots of 15 μl of each fraction were diluted 1in 2 in SDS sample buffer and electrophoresed in 13% w/v polyacrylamidegels in a Mini-Protean II system (BioRad) and stained with CoomassieBrilliant Blue. The positions of the molecular weight markers are shown.

FIG. 11B is a graphical representation showing monitoring of mLBPamounts by direct ¹²⁵I-mLIF binding assays of elution fractions fromhLIF-Affigel-10 column.

FIG. 12 is a photographic representation showing deglycosylation of mLBPusing N-glycanase. Aliquots of ¹²⁵I-mLBP in 50 mM sodium phosphate pH7.5were incubated in the presence of 0.25 units of N-glycanase (Genzyme) inthe same buffer (lanes a and b), buffer alone (lane c) or 0.25 units ofN-glycanase, 0.1% w/v SDS and 1% v/v 2-mercaptoethanol in the samebuffer (lanes d and e) at 37° C. for 24 hours. The incubation mixtureswere diluted 1 in 2 in SDS sample buffer and electrophoresed in 10% w/vpolyacrylamide gels in a Mini-Protean II system (BioRad), stained withCoomassie Brilliant Blue, dried and exposed to a phosphorimager screen(Molecular Dynamics) for 24 hours. Results were analysed usingImagequant version 3 (molecular Dynamics) software. The positions of themolecular weight markers are shown.

EXAMPLE 1 1. Materials and Methods

Collection of Mouse Sera

Blood from C57BL/6J, C3H/HeJ, CBA f/Ca H, DBA/2J and BALB/C/An Bradleymice was collected by exsanguination. Red cells were separated from theserum by centrifugation at 3000 g for 10 min, and the serum stored at−20° C. for up to 6 months.

Affinity Chromatography

Leukaemia Inhibitory Factor p-aminobenzamidoethyl-Sepharose 4B(LIF-pABAE Sepharose 4B) was prepared according to the method ofCuatrecasas and Anfinsen². Briefly, 8.4 g CNBr-Activated Sepharose 4B(Pharmacia, Uppsala) was washed in 1 mM HCl, reacted with an equalvolume of 2M ethylene diamine at pH10 for 16 hr at 4° C. Thep-nitrobenzamido-ethyl Sepharose 4B was washed extensively with 50% v/vdimethyl formamide, then reduced with 0.2M sodium dithionite in 0.5Msodium bicarbonate pH8.5 for 60 min. at 40° C. The p-aminobenzamidoethylSepharose 4B was washed in 0.5M HCl then diazotised with 0.1M sodiumnitrite for 7 min at 4° C. Aliquots of 7 ml of this diazonium-Sepharosederivative were then reacted with 8 mg each of E. coli-derived murineLIF³ and chicken ovalbumin (Sigma, MO) as a non-specific filler proteinin 10 ml 0.2M sodium tetraborate pH9.2 for 16 hr at 4° C. TheLIF-pABAE-Sepharose 4B was washed extensively with 20 mM sodiumphosphate pH7.4 containing l50 mM sodium chloride (PBS) and 6Mguanidine-HCl in PBS before use.

Aliquots of 50 ml mouse serum or pooled fractions containing LIF-bindingprotein (LBP) activity in PBS were applied to a 13×0.7 cm column ofLIF-pABAE-Sepharose 4B equilibrated in PBS (this and all succeedingbuffers contained 0.02% v/v Tween 20 and 0.02% w/v sodium azide) andeluted with 20 ml equilibration buffer followed by a 15 ml lineargradient to 6M guanidine-HCl in the same buffer. Fractions of 2.5 mlwere collected at a flow rate of 0.5 ml/min and exchanged wherenecessary into equilibration buffer using a prepacked Sephadex G-25column (PD10, Pharmacia, Uppsala).

Anion-exchange Chromatography

Fractions from affinity chromatography containing LIF-binding activitywere pooled and diluted 20-fold with 20 mM Tris pH7.0, then applied to aMono-Q HR 5/5 (Pharmacia, Uppsala) column equilibrated in the samebuffer. Elution was carried out using 25 ml of equilibration buffer,followed by a 30 ml linear gradient to 1M NaCl in equilibration buffer.The 0.5 ml fractions were collected at a flow rate of 0.5 ml/min.

Size-exclusion Chromatography

Fractions from anion-exchange chromatography containing LIF-bindingactivity were pooled and concentrated to 100 μl using a Centricon-10microconcentrator (Amicon, MA). The sample was injected onto aSuperose-12 10/30 (Pharmacia, Uppsala) column equilibrated in PBS, andelution was carried out isocratically using the same buffer. The 0.2 mlfractions were collected at a flow rate of 0.2 ml/min.

Preparative Native Polyacrylamide Gel Electrophoresis

Fractions from size-exclusion chromatography containing LIF-bindingactivity were diluted 2-fold in 0.062M Tris pH6.8, 12.5% v/v glycerol,0.02% w/v bromophenol blue, applied to a 20 ml 0.375M Tris pH8.8, 7.5%w/v polyacrylamide/0.2% w/v Bis separating gel with a 10 ml 0.125M TrispH6.8, 4% w/v acrylamide/0.11% w/v stacking gel, containing no sodiumdodecyl sulfate (NaDodSO₄). The sample was electrophoresed in a Model491 Prep Cell (BioRad, CA) using a 0.025M Tris, 0.19M glycine bufferpH8.3 as an electrode buffer at 40 mA for approximately 6 hr. Fractionsof 2.5 ml were collected at a flow rate of 1 ml/min after the elution ofthe bromophenol blue dye front from the gel.

Analytical Sodium Dodecyl Sulfate Polyacrylamide Gel Eelectrophoresis

NaDodSO₄-PAGE was performed according to the method of Laemmli⁴.Aliquots of samples to be analysed were diluted in NaDodSO₄ samplebuffer and electrophoresed in 12.5% w/v polyacrylamide gels in aMini-Protean II system (BioRad, CA) and silver-stained⁵.

Iodination of LIF

Amounts of 1-2 μg recombinant murine or human LIF produced in E. coliwere iodinated and purified as previously described⁽³⁾. The iodinatedmaterials retained biological activity and had specific activities of3-5×10⁵ cpm/pmole for ¹²⁵I-mLIF and 6-10×10⁵ cpm/pmole for ¹²⁵I-hLIF.

Binding Assays

Aliquots of samples containing the LIF-binding activity were added toEppendorf tubes containing 10 μl ¹²⁵I-LIF (5-10×10⁴ cpm) and 50-100 μlconcanavalin-A Sepharose (Pharmacia, Uppsala) (diluted four-fold in 100mM sodium acetate pH6.0 containing 1 mM each MgCl₂, MnCl₂ and CaCl₂).Non-specific binding was determined from incubations containing inaddition 10 μl of 50 μg/ml unlabelled LIF. Assay tubes were incubated atroom temperature for 16 hr with agitation. Bound and free label wereseparated by resuspending the incubation mixture, layering it over 150μl fetal calf serum (FCS) in a tapered microcentrifuge tube and spinningat 13,000 rpm for 1 min. The tip of the tube, containing the Sepharosepellet, was then cut off using a scalpel blade and both the pellet andsupernatant were counted for 1 min in a gamma counter (Packard CrystalMultidetector, Packard Instruments).

For competitive binding experiments, LBP and Concanavalin-A Sepharosewere incubated with between 1×10⁻⁴ and 2×10⁴ ng unlabelled ligand and afixed concentration (2 nM) of labelled ligand and then processed asdescribed above.

Scatchard Analyses

3T3-L1 cells were maintained as previously described⁶ and harvestedusing Hepes buffered RPMI medium with 10% v/v fetal calf serum (HRF)containing 40 mM EDTA and 200 μg/ml chondroitin sulfate. Cellsresuspended in 60 μl HRF were placed in Falcon 2054 tubes (BectonDickinson, NJ) with 10 μl ¹²⁵I-mLIF, then incubated at 4° C. for 3 hr.Non-specific binding was determined as above. Mouse liver membranes wereprepared essentially as described⁷, solubilised in 1% v/v Triton X-100(Pierce, IL) and assayed as for LBP. Saturation binding experiments werecarried out and Scatchard analysis of the binding isotherm wasdetermined using the curve-fitting program, LIGAND⁸, after correctionfor the bindable fraction of radioligand as previously described⁽³⁾.Scatchard analysis allows the derivation of the affinity of a labelledligand for its binding site and the concentration of these bindingsites, thereby permitting the quantitation of LBP in samples of knownvolume, assuming one binding site per LBP molecule and a molecularweight of 90 kDa for LBP.

Bioassay

Samples for bioassay were exchanged into normal saline using prepackedSephadex G25M (PD10 columns, Pharmacia, Uppsala), concentrated usingCentricon-10 microconcentrators (Amicon, MA) and sterilised by passagethrough a 0.45 μm filter (Millipore, MA). M1 differentiation assays wereperformed as previously described⁹.

Protein Assay

Protein was estimated by the method of Bradford⁽¹³⁾ using a Coommassieblue based reagent (Pierce, IL) according to the manufacturersinstructions.

Amino Acid Sequence Analysis

Amino acid sequence analysis was carried out by Edman degradation ofproteins blotted onto a polyvinylidene difluoride (PVDF) membrane aspreviously described¹⁰.

2. Analysis of LIF Binding Protein

Identification of LIF-binding Activity in Mouse Serum

LIF-binding protein (LBP) was detected in normal mouse sera by itsability to inhibit the binding of ¹²⁵I-mLIF to a neutralising monoclonalantibody in a competition radioimmunoassay or to its cellular receptoron 3T3-L1 cells in a competition radioreceptor assay. It was detected byeither of these assays in normal mouse sera at a dilution of 1/4 to 1/8,but at a 20 to 30-fold greater dilution in the sera of pregnant mice anda 2-4 fold lower dilution in the sera of neonatal mice (Table 2).Because LIF is a very basic protein, it was important to determinewhether this LIF-binding activity was due to a non-specific associationof LIF with an acidic serum protein, however a variety of other basicproteins, including lysozyme, α-chymotrypsinogen-A and cytochrome-C,were unable to compete for ¹²⁵I-mLIF binding to LBP. In addition, otheracute-phase proteins, such as IL-1 and IL-6, were also unable to competefor binding.

Direct binding of 125I-mLIF to LBP was detected in two different ways.By size-exclusion chromatography, ¹²⁵I-mLIF exhibits a molecular weightof approximately 20 kDa. When ¹²⁵I-mLIF was chromatographed in thepresence of normal mouse serum, under non-dissociating conditions, alabelled complex was detected with an apparent molecular weight of 110kDa (FIG. 1), signifying that the LIF-binding protein in serum had amolecular weight of about 90 kDa Concanavalin-A Sepharose wasdemonstrated to have the ability to precipitate the ¹²⁵I-mLIF-LBPcomplex from serum, implying that LBP was a glycoprotein, since therecombinant LIF labelled for these studies was produced in E. coli andso contained no carbohydrate groups. This property was used to separatebound from free ¹²⁵I-LIF as recently described for solubilised receptorassays¹¹.

Isolation of LBP

The purification of mLBP from normal mouse sera was achieved bysequential fractionation using affinity chromatography on an immobilisedLIF column, anion exchange chromatography, size exclusion chromatographyand preparative native polyacrylamide gel electrophoresis (PAGE), asdetailed in the methods section, and monitoring LBP by direct ¹²⁵I-mLIFbinding assays.

LBP was found to be a very unstable protein that was completelyinactivated by acidic conditions and in the presence of many of thechemicals usually used in protein purification procedures, such asacetonitrile, methanol, NaDodSO₄ and ammonium sulfate. This proved themajor difficulty in the isolation of mLBP as it precluded the use ofmany high-resolution purification techniques, and may be the basis forthe relatively low yields obtained during fractionation (Table 3).

Chemical modification of lysine residues in the LIF molecule has beenshown to destroy its biological activity whereas iodination of tyrosineresidues is not detrimental to LIF activity⁽³⁾. A method of linkingprotein to Sepharose beads through tyrosine residues was thereforechosen to ensure that the LIF molecule would be active when attached toan affinity column matrix. Affinity chromatography (FIG. 2A) on anmLIF-pABAE Sepharose 4B affinity matrix removed a large proportion ofthe contaminating serum proteins, although the guanidine used as aneluent had some denaturing effects on LBP activity. A second round ofaffinity chromatography resulted in a significant additionalpurification. In contrast, LBP did not bind to a control ovalbumin-pABAESepharose 4B column. Anion exchange (FIG. 2B) and size exclusionchromatography (FIG. 2C) confirmed the acidic nature of the protein andits molecular weight of approximately 90 kDa, and achieved a further1.6-fold purification. Analytical NaDodSO₄-PAGE of fractions from thesize exclusion column revealed two bands that were barely separated fromeach other on a 12.5% w/v polyacrylamide gel, but were well separatedfrom other contaminating bands. Both had an apparent molecular weight ofapproximately 90 kDa and their distributions in size exclusion columnfractions and native preparative gel fractions as determined by silverstaining of analytical NaDodSO₄-PAGE gels exactly matched that of LBPactivity by direct ¹²⁵I-mLIF binding assay. The two peak fractions wereelectrophoresed on a 7% w/v NaDodSO₄-PAGE gel, blotted onto a PVDFmembrane and the two bands of interest were subjected to N-terminalsequencing. The major band had a slightly lower molecular weight andyielded amino acid sequence at the 5 picomole level (23 amino acidresidues), while the higher molecular weight band yielded sequence atthe 3 picomole level (8 amino acid residues). Both revealed anN-terminal sequence consistent with the following sequence:Gly-Val-Gln-Asp-Leu-Lys-Cys-Thr-Thr-Asn-Asn-Met-Arg-Val-Trp-Asp-Cys-Thr-Trp-Pro-Ala-Pro-Leu(SEQ ID No. 1). The two forms of mLBP were not seen in every preparationand were presumed to be glycosylation variants, although the C-terminusof mLBP is yet to be identified and differences at this end of themolecule could account for the slight size heterogeneity.

Preparative gel electrophoresis using native conditions (FIG. 3A)allowed high resolution separation of LBP from contaminating proteinswith only two major bands of 90 and 67 kDa revealed by analyticalNaDodSO₄-PAGE after pooling active fractions from this step (FIG. 3B).Fractions pooled from this step were used in assays to determine thebinding characteristics of LBP.

Binding Properties of LBP

The specific binding of ¹²⁵I-mLIF to purified LBP is shown in FIG. 4AScatchard analysis of the saturation binding isotherm of ¹²⁵I-mLIF tonormal mouse sera or purified LBP showed that LBP contained a singleclass of mLIF binding site (K_(D) 500 pM-3 nM) and indicated that normalmouse serum contains approximately 1-5 μg/ml of LBP (FIG. 4B). Theaffinity of LBP for mLIF was comparable to that of the low affinity LIFreceptor solubilised by detergent from mouse liver membranes (K_(D)=680pM) and was significantly lower than that of the high affinity cellularreceptor on 3T3-L1 cells (K_(D)=57 pM) (FIG. 4C).

Although hLIF was able to bind to mLBP, its binding characteristics wererather different. Unlabelled mLIF and hLIF showed a similar ability tocompete with ¹²⁵I-mLIF for binding to mLBP (FIG. 5A), but mLIF wasconsistently 10,000-fold less effective than hLIF in competing with¹²⁵l-hLIF for binding to mLBP (FIG. 5B).

Blocking Activity of LBP

When affinity purified mLBP was combined with mouse LIF in cultures ofmurine M1 myeloid leukemic cells, it blocked the ability of LIF toinduce differentiation of M1 colonies in a dose dependent manner (50%inhibition of 90 U/ml LIF at 62-125 ng/ml LBP). Intermediate doses ofmLIF (stimulating 50% differentiation of M1 colonies) revealedinhibition by high doses of LBP, but possibly augmentation at low dosesof LBP (2-4 ng/ml) (FIG. 6A). The presence of 125 ng/ml LBP in theculture was able to shift a mLIF titration curve 2-fold towards highermLIF doses (FIG. 6B).

FIG. 7 shows the blocking of induction of mLIF and hLIF stimulatedcolonies of M1 leukaemic cells by normal mouse serum. In 1 ml culturesof 300 murine M1 myeloid leukaemic cells, 0.1 ml of serum from normaladult (8 week) C57BL mice was able to block the differentiation-inducingeffects after 7 days of incubation of 100 Units of purified recombinantE. coli-derived murine LIF (50% blocking with a serum dilution of 1:8)(FIG. 7A). This serum had a greater capacity to block the effects of 800Units of purified recombinant E. coli-derived human LIF (50% blockingwith a serum dilution of 1:512). A similar heightened activity inblocking human LIF was seen using purified recombinant human LIFexpressed in E. coli, CHO cells or in yeast (FIG. 7B).

EXAMPLE 2 m-LIF and hLIF Binding to mLBP

FIG. 8 is a graphical representation showing specific binding of¹²⁵I-m-LIF (A) and ¹²⁵I-LIF (B) to mLBP immobilised onto 96 well PVCplates. The results show that mLIF immobilised to a solid supportprovides a ligand for capturing hLIF in a sample. FIG. 8 shows that hLIFbound to the mLBP at least 20-30 fold better than mLIF as measured byspecific binding. Apart from the therapeutic implications on such afinding, these results identify mLBP as a suitable ligand for assayingthe presence of hLIF in a biological sample.

EXAMPLE 3

Fusion LBP Polypeptides

Administration of a mouse protein to a human subject may induce anantigenic immune response with therapeutically undesirable consequences.In order to avoid or reduce this potential problem, a protein that isantigenically very similar to a human LBP, but which retains thehigh-affinity for hLIF that is the particular property of mLBP isconstructed. The method used is similar to the method the inventors havepreviously used to map the site on the hLIF molecule that confers bothbinding to the hLIF receptor α-chain and the unusual high-affinitybinding to the mouse LIF receptor α-chain (mLBP). Using a mLIF molecularframework, the inventors constructed a series of mouse-human LIFchimaeric molecules in order to determine the minimum number of hLIPamino acid residues that it is necessary to substitute into the mLIFsequence in order to create a molecule that has the properties that arepeculiar to hLIF (see FIG. 9).

By constructing a series of mouse-human LBP chimaeric molecules usingrecombinant DNA technology, the minimum number of mLBP amino acidresidues is determined that it is necessary to substitute into a hLBP(soluble human LIF receptor α-chain) sequence in order to create amolecule that binds to hLIF with high affinity, as mLBP does, ratherthan a molecule that binds hLIF with low affinity, as does hLBP (FIG.10). Recombinant mLBP molecule is anticipated to have identical bindingand inhibition properties to the native form of mLBP isolated fromnormal mouse serum, as all forms of the mouse LIF receptor tested,whether cellular or soluble, have shared the binding properties of mLBP.The ultimate aim is to synthesize a recombinant molecule that is basedon the hLIF receptor α-chain amino acid sequence, but is C-terminallytruncated so that it is a soluble rather than cell membrane-boundmolecule, is of the minimum size that retains LIF-binding properties andis easily expressed in large quantities. In addition, this molecule willcontain approximately 5-15 amino acid residues that are substituted forthe amino acid residue that is in the identical position in the mouseLIF receptor α-chain amino acid sequence. These substitutions willspecifically induce the hLBP molecule to bind hLIF with high affinity,and thus act as an effective hLIF antagonist in vitro or in vivo,however, these few amino acid substitutions would be unlikely to causethe human LBP molecule to become antigenic to the human immune system invivo.

EXAMPLE 4

Digestion of mLBP with N-glycanase

N-terminal amino acid sequencing of mLBP suggests that it is a truncatedform of the cellular LIF receptor α-chain. It is possible to estimatethe size of a protein by SDS-PAGE, and correlate this with the predictedmolecular weight of the protein, calculated by adding up the individualmolecular weights of all the amino acid residues in the amino acidsequence. The mLBP isolated from mouse serum has the ability to bind toa carbohydrate-binding lectin, Concanavalin-A, and so must be aglycosylated molecule. Techniques are not readily available to estimatethe proportion of carbohydrate versus protein in a glycosylated protein,so the apparent 90-95 kDa molecular weight of mLBP, as estimated bySDS-PAGE, cannot be correlated with the amino acid sequence.

A second form of mLBP has become apparent in purified preparations ofmLBP over time, and is presumed to be formed by proteolytic degradation.This form of mLBP is capable of binding both mLIF and hLIF, and has amolecular weight of approximately 65 kDa as estimated by SDS-PAGE (FIG.11A,B).

In order to estimate the position of the C-terminus of both the 90-95kDa and 65 kDa forms of mLBP, preparations containing both these formswere subjected to digestion by N-glycanase, an enzyme that specificallycleaves the bond between an asparagine residue and an N-linkedcarbohydrate. Digestion was allowed to go to completion and themolecular weights of the deglycosylated proteins were estimated bySDS-PAGE (FIG. 12). The two bands produced by N-glycanase digestion hadmolecular weights of approximately 63 kDa and 50 kDa, respectively.These bands corresponded to the molecular weights of the protein coresof the two forms of mLBP, without the N-linked carbohydrate, and socould be correlated with the amino acid sequence.

The cellular mLIF receptor comprises two haemopoietin domains, threefibronectin III-like domains, a transmembrane domain and a cytoplasmicdomain, and has a glycosylated molecular weight of 190 kDa⁽¹²⁾. Themolecular weight of the reported form of the soluble mLIF receptor whichconsists of two haemopoietin domains and two out of three fibronectinIII-like domains is 130 kDa when glycosylated ⁽¹²⁾ and 75 kDa for thecore protein. The predicted molecular weight for the core protein forconsisting of two haemopoietin domains and one out of three fibronectinIII-like domains is 63 kDa, while for the two haemopoietin domainsalone, the predicted molecular weight is 53 kDa. These molecular weightscorrespond well to the molecular weights of the two deglycosylated formsof mLBP as estimated by SDS-PAGE, which suggests that a variety of formsof mLBP are created by sequential proteolytic removal of fibronectinIII-like domains and that each of these forms retains the bindingactivity described for mLBP.

EXAMPLE 5 Determination of the Amino Acid Sequence of the C-terminus ofmLBP

Further information about the C-terminal amino acid sequence of mLBP isobtainable by sequencing proteolytically-produced peptide fragments ofmLBP. In order that sequence data can be gained from the differentmolecular weight forms of mLBP using as little protein as possible, theproteolytic digestion is carried out within an SDS-PAGE gel and thepeptides extracted from the gel and separated by reversed-phase HPLC formicrosequence analysis.

Briefly, the Coomassie Blue stained band of the correct molecular weightis cut out of the gel and destained with 50% v/v n-propanol containing3% w/v SDS. The gel slice is then washed extensively with water anddried by centrifugal lyophilization. The gel slice is then rehydrated in100 μl 0.1M NaHCO₃ containing 0.02% v/v Tween-20 and 1-2 μg of protease(for example, trypsin or LysC). Peptides are then extracted from the gelslices with 100 μl 1% v/v trifluoroacetic acid (TFA) for 4 hours, 100 μl70% v/v TFA for 4 hours, 100 μl 70% v/v TFA for 16 hours and 100 μl 50%v/v TFA/50% acetonitrile for two times 4 hours. The combined eluents areevaporated to near dryness then diluted to 1 ml with 0.1% v/v TFA. Thepeptides are then separated by reversed-phase HPLC and subjected toamino acid sequence analysis.

TABLE 2 LEVELS OF LBP IN SERUM¹ DILUTION GIVING 50% LBP AGE INHIBITIONCONC¹ SERUM TYPE STRAIN SEX (wks) IN RIA (μg/ml) MOUSE ADULT MIXED MIXEDN/A³ 1/4-1/8 1 ″ C57 M 6 1/4 1 ″ C57 F 6 1/4 1 ″ CBA F 12  1/4 1NEONATAL CBA F 1 1/2 0.5 ″ CBA M 1 1/1 0.25 ″ CBA M 2 1/4 1 PREGNANT CBA² F 12   1/128 32 HUMAN N/A³ MIXED N/A³ N/D⁴ 0 RAT N/A³ MIXED N/A³1/4 1 ¹Levels of LBP detected in serum expressed as the dilution ofserum required to inhibit 50% of specific binding of ¹²⁵I-mLIF to aneutralising monoclonal antibody in a competition RIA. The number of¹²⁵I-mLIF binding sites in normal mouse serum was determined byScatchard analysis to be 1 μg/ml and used as a standard to convert 50%inhibition in RIA to LBP concentration. ²14 days pregnant ³NotApplicable ⁴Not Detectable

TABLE 3 REPRESENTATIVE PURIFICATION OF LBP FROM NORMAL MOUSE SERUM¹TOTAL FRACTIONATION VOL TOTAL PROTEIN YIELD P² PROCEDURE (ml) LBP (μg)(μg) (%) (fold) Normal Serum 85 102 2.24 × 10⁶ — — LIF Sepharose 1 78 411.54 × 10⁴ 39.8  58 LIF Sepharose 2 38 18 2.73 × 10³ 19.4 147 AnionExchange 8.9 18 2.45 × 10³ 19.9 158 Size Exclusion 3.2 6.0 573 7.8 232Native Gel 0.8 1.4 11.6 1.4 2620  ¹Representative purification of LBPfrom normal mouse serum. Total LBP was calculated from the concentrationof ¹²⁵I-mLIF binding sites derived from Scatchard analysis, assuming onebinding site per LBP molecule and a molecular weight of 90 kDa for LBP.²Purification (fold)

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

REFERENCES

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4. Laemmli, U. K. Nature 227: 680-685, 1970.

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6. Green, H. and Kehinde, O. Cell 1: 113-116, 1974.

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8. Munson, P. J. and Rodbard, D. Anal. Biochem. 107: 307-310, 1980.

9. Metcalf, D., Hilton, D. J. and Nicola, N. A. Leukaemia 2: 216-222,1990.

10. Ward, L. D., Hong, J., Whitehead, R. H. and Simpson, R. J.Electrophoresis 11: 883-891, 1990.

11. Nicola, N. A. and Cary, D. A. Growth Factors 6: 119-129, 1992.

12. Gearing, D. P. Thut, C. J. VandenBos, T., Gimpel, S. D., Delaney, P.B., King, J., Price, V., Cosman, D., and Beckman, M. P. EMBO J. 10:2839-2848, 1991

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1 23 amino acids amino acid single linear protein 1 Gly Val Gln Asp LeuLys Cys Thr Thr Asn Asn Met Arg Val Trp Asp 1 5 10 15 Cys Thr Trp ProAla Pro Leu 20

What is claimed is:
 1. A method of inhibiting the activity of humnan LIFto induce differentiation of human M1 myeloid Leukaemic cells comprisingadministering to a human an effective amount of mouse LeukaemiaInhibitory Factor (LCF)-binding protein (LBP) in soluble form, said LBPexhibiting at least 20-fold higher binding affinity for human LIFcompared to the binding affinity for mouse LIF.
 2. A method ofinhibiting the activity of human LIF comprising administering to a humanan effective amount of a soluble mouse Leukaemia InhibitoryFactor-binding protein (LBP), wherein said mouse LBP exhibits at least20-fold higher binding affinity for human LIF compared to the bindingaffinity for mouse LIF.